A rare episode of regeneration of aspen (Populus tremuloides Michx.) by seeds occurred in Yellowstone National Park (YNP), Wyoming, USA, following extensive fires that occurred in 1988. In 1997, we sampled 410 aspen seedlings from 23 local populations distributed widely across YNP to determine how genetic diversity varies with elevation, substrate, plant competition, ungulate browsing, and geographical location. We employed 132 randomly amplified polymorphic DNA (RAPD) markers based on six primers to show genetic relationships within and among the postfire aspen seedling populations. Measures of genetic variation, including estimates of percentage polymorphic loci, expected heterozygosity, and Nei’s FST, indicated that most of the variation occurred within rather than among local populations. There was no indication of geographical differentiation among sampled populations based on hierarchal estimates of Nei’s FST, neighbour-joining, or correlations between genetic distance and geographical distance. Even genetically distant populations shared nearly 90% of the same markers. Within plots, the amount of genetic variation decreased slightly in response to increased percentage vegetative cover, mean seedling basal diameter, and mean seedling height. Geological substrate, density of lodgepole pine (Pinus contorta var. latifolia Dougl.) seedlings, browsing intensity, and elevation were not significantly related to levels of genetic variation within the seedling plots. These data suggest that genetic variation and geographical structure among seedling populations may occur over time as the transition from seedling-dominated stands to clone-dominated stands occurs.